A crucial role of purine nucleoside metabolism in immune function and in regulating de novo purine biosynthesis has been identified by the association of immune deficiency in patients lacking adenosine deaminase and purine nucleoside phosphorylase and the finding of purine overproduction by the latter. The objectives of my proposal are: 1) To determine the basis for the toxicity of adenosine to lymphoid cells which may be the basis for immune dysfunction in adenosine deaminase deficiency. 2) To determine the cause of purine overproduction in individuals deficient in nucleoside phosphorylase, and to better define normal regulation of the rate of de novo purine synthesis in intact cells. 3) To clarify the regulation of the distal branchpoint in the de novo purine synthetic pathway and the role of adenosine kinase and other enzymes of purine salvage in this regulation. A cultured human lymphoblast line will be used as a model, and mutants will be selected from it in order to pursue these objectives. Specific studies will involve: a) Characterization of differences in the biochemical effects of adenosine on normal lymphoblasts and mutants resistant to the toxicity of adenosine. b) Characterization and purification of a cytoplasmic adenosine binding protein to determine its possible role in mediating the effects of adenosine. c) Selection of nucleoside phosphorylase deficient mutants and study of the effects of this deficiency on parameters which may regulate de novo purine synthesis. d) Selection of mutants with a feedback-resistant PRPP amidotransferase (the rate limiting enzyme of de novo purine synthesis) and comparisons of normal and mutant enzymes. e) Study of the effects of these mutations and other factors on regulation of the relative amount of newly synthesized IMP converted to adenine and guanine nucleotides during the cell cycle and in transitions between resting stage and active cell growth.